Interleukin-4-mediated 15-lipoxygenase-1 trans-activation requires UTX recruitment and H3K27me3 demethylation at the promoter in A549 cells

Abstract

Arachidonate 15-lipoxygenase-1 (ALOX15) oxygenates polyunsaturated fatty acids and bio-membranes, generating multiple lipid signalling mediators involved in inflammation. Several lines of evidence indicate that ALOX15 activation in the respiratory tract contributes to asthma progression. Recent experimental data reveals that histone modification at the promoter plays a critical role in ALOX15 gene transcription. In the present study, we examined the status of histone H3 trimethyl-lysine 27 (H3K27me3) at the ALOX15 promoter by chromatin immunoprecipitation assay in human lung epithelial carcinoma A549 cells incubated with or without interleukin (IL)-4. We identified demethylation of H3K27me3 at the ALOX15 promoter after IL-4 treatment. Furthermore, we found that the H3K27me2/3-specific demethylase, ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX), mediates the H3K27me3 demethylation during ALOX15 transcriptional activation. When UTX expression was knocked down using siRNA, IL-4-mediated H3K27me3 demethylation and ALOX15 induction were significantly attenuated. The critical role of UTX in ALOX15 expression was confirmed in human monocytes and the Hodgkin lymphoma (HL) cell line L1236, but was in these cells not related to H3K27me3-demethylase activity. These results demonstrate that UTX is implicated in IL-4 mediated transcriptional activation of the ALOX15 gene.

Figure 1. IL-4 induces ALOX15 expression in A549 cells.

A549 cells were cultured in RPMI 1640 medium containing 50/mL IL-4 for indicated time points. (A) Total RNA was purified and ALOX15 and UTX mRNA was measured by qRT-PCR; (B) and the protein level of ALOX15 was measured by Western blot; (C) the dose effects of IL-4 on ALOX15 induction was determined by qRT-PCR. The western blot results represent two independent experiments, and the error bars represent standard error mean of three independent qRT-PCR experiments.

Figure 2. IL-4 mediates recruitment of UTX and histone modification at the ALOX15 promoter.

(A) Schematic presentation of the ALOX15 promoter and locations (relative to ATG) of PCR primers used for ChIP assay; (B) Anti-H3K27me3 ChIP assay covering the indicated ALOX15 promoter regions was performed in A549 cells after IL-4 stimulation; (C) Anti-UTX ChIP assay was performed on the indicated ALOX15 promoter regions in A549 cells after IL-4 stimulation. The GAPDH primers were used as a negative control. Bars represent mean value of ChIP signals normalized to 1% input. Error bars represent standard error mean of three independent experiments. *p<0.05; ** p<0.01; *** p<0.001.

Figure 3. Lysine (K)-specific demethylase UTX is required for ALOX15 induction in A549 cells.

UTX specific siRNA was transfected 72-4 treatment, and total RNA and protein were purified, followed by qRT-PCR and Western blot, the mRNA (A) and protein level (B) of UTX was measured upon UTX depletion followed by IL-4 stimulation. (C) The status of H3K27me3 at ALOX15 promoter region 3(see figure? 2) was verified upon UTX depletion followed by IL-4 stimulation; (D) the effect of UTX depletion on IL-4-induced ALOX15 expression was measured by qRT-PCR. All qRT-PCRs used GAPDH as loading control and the relative expression levels were calculated as the values relative to those of the calibrator samples (untreated sample). β-Actin was used as a loading control for all western blots. qRT-PCR data is shown as “fold induction” relative to that in control cells. Error bars represent standard error mean of three independent experiments. *p<0.05; ** p<0.01; *** p<0.001.

Figure 4. The expression of ALOX15 in human peripheral monocytes and HL-derived L1236 cells requires UTX.

Human monocytes were treated as indicated. Quantitative RT-PCR analysis and Western blot were performed. (A) The dose response effect of IL-4 (12 hours) on ALOX15 mRNA induction; (B) the time course of ALOX15 transcription in response to IL-4 (50 ng/ml) stimulation; (C) the dose response effect of IL-4 (48 hours) on ALOX15 protein expression; (D) the time course of ALOX15 expression in response to IL-4 (50 ng/ml) stimulation. (E, upper-panel) Monocytes were transduced with UTX-specific or scrambled short hairpin RNA by means of a lentiviral vector, and simultaneously supplied with IL-4 (50 ng/ml) containing medium. The monocytes were harvested and quantitative RT-PCR analyses performed after five days culture. (E, lower panel) Monocytes were stimulated with IL-4 (50 ng/ml) for 24 hours and thereafter ChIP assay was carried out by using anti-H3K27me3 and anti-UTX antibodies, and quantitative PCR was performed using the primers covering the ALOX15 promoter region 3 (see figure? 2). (F, upper-panel) L1236 cells were transduced with either UTX-specific or scrambled short hairpin RNA by using lentiviral vector, and cells were harvest, total RNA were purified and quantitative RT-PCR analyses were performed after 7-days since transduction. (F, lower panel) L1236 cells were analyzed by ChIP assay using anti-H3K27me3 and anti-UTX antibodies, and followed by PCR using the primers covering the ALOX15 promoter region 3 (see Figure? 2). Rabbit IgG was used in ChIP as a negative control. Quantitative RT-PCR data is shown as “fold induction” relative to that in controls. Error bars represent standard error mean of three independent experiments. The Western blot and ChIP assay results represent three independent experiments. *p<0.05; ** p<0.01; *** p<0.001.